Purification and characterization of barley-aleurone xylanase

Xylanase (-1,4-D-xylan xylanohydrolase; EC 3.2.1.8) from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was purified and characterized. Purification was by preparative isoelectric focusing and a Sephadex G-200 column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight (Mr)=34000 daltons. The isoelectric point of the enzyme was 4.6. The enzyme had maximum activity on xylan at pH 5.5 and at 35° C. It was most stable between pH 5 and 6 and at temperatures between 0 and 4° C. The K_m was 0.86 mg xylan·ml^-1.Abbreviations GA₃ gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Arabinogalactan protein and wall-associated kinase in a plasmalemmal reticulum with specialized vertices

Summary Arabinogalactan protein and wall-associated kinase (WAK) are suspected to be regulatory players at the interface between cytoplasm and cell wall. Both WAK(s) and arabinogalactan shown likely to represent arabinogalactan protein(s) have been visualized there with computational optical-sectioning microscopy. The arabinogalactan occurs in a polyhedral array at the external face of the cell membrane. WAK, and other proteins as yet unidentified, appear to fasten the membrane to the wall at vertices of the array. Evidence is presented that the array bears an important part of the mechanical stress experienced by the membrane, and it is speculated that the architectural organization of arabinogalactan protein, WAK, and other components of the array is critical for coordination of endomembrane activities, growth, and differentiation. The array has been named the plasmalemmal reticulum.Abbreviations AGP arabinogalactan protein - (-D-Glc)₃ -D-glucosyl Yariv phenylglycoside - (-D-Man)₃ -D-mannosyl Yariv phenylglycoside - SDS-AGE sodium dodecyl sulfate-agarose gel electrophoresis - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - WAK wall-associated kinase     

Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin

The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml^-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[³H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (M_r) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an M_r of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[³H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([³H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an M_r of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [³H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC [(4-azidobenzoyl)-ethylenediamine]-fusicoccin - ABE-NHS (4-azidobenzoyl)-N-hydroxysuccinimide ester - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-norfusicoccin-8-alcohol - MAB monoclonal antibody - Mega-9(10) nonanoyl(decanoyl)-N-methylglucamide - M_r apparent molecular mass - PMSF phenylmethyl-sulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Kiwifruit β-galactosidase: Isolation and activity against specific fruit cell-wall polysaccharides

A -galactosidase (EC 3.2.1.23) capable of degrading a number of fruit cell-wall polysaccharides in vitro, was isolated from ripening kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson cv. Hayward). The enzyme has a molecular weight of approximately 60 kDa by gel permeation and consists of several basic isoforms. Several polypeptides were enriched during purification, with 33-, 46- and 67-kDa bands being predominant after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme against p-nitrophenyl--d-galactopyranoside was at pH 3.2, but against a galactan purified from kiwifruit cell walls, it was at pH 4.9. The enzyme was specific for galactosyl residues in the -configuration, releasing galactose from a variety of kiwifruit cell-wall polysaccharide fractions including cell wall material, Na₂CO₃-soluble pectin, high-molecular-weight galactan, xyloglucan, and galactoglucomannan. A galactosylated glucuronomannan found throughout the kiwifruit plant was also a substrate for the enzyme. The results indicate that the enzyme attacks the non-reducing end of galactose side chains, cleaving single galactose residues which may be attached to the 2, 3, 4, or 6 position of the aglycone. Activity of the enzyme in-vitro was too low to account for the total loss of galactose from the cell walls during ripening. If the -galactosidase of this study is solely responsible for the removal of galactose from the cell wall during ripening then its in-vivo activity must be much greater than that observed in-vitro.Abbreviations CWM cell wall material - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We thank Bronwyn Culling and Teresa Wegrzyn for assistance and acknowledge a contribution towards the cost of the research from the New Zealand Kiwifruit Marketing Board.     

Identification and characterization of major bovine serum tyrosine-O-sulfate-binding protein as a complement factor H

A major tyrosine-O-sulfate (TyrS)-binding protein present in bovine serum was purified to electrophoretic homogeneity using a combination of TyrS-Affi-Gel 10 affinity chromatographyy, DEAE-Bio-Gel A ion-exchange chromatography, and hydroxylapatite chromatography. The purified TyrS-binding protein migrated as doublet protein bands with apparent molecular weights of ca. 160, 000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. N-termini of the two forms of purified TyrS-binding protein contain most likely identical sequence for the first fifteen amino acids residues, which displays a high degree of homology to those of human and mouse complement factor H. Furthermore, the purified TyrS-binding protein exhibited immunologic cross-reactivity with anti-human complement factor H. These results indicate the identity of the purified TyrS-binding protein being bovine complement factor H. The two forms of the purified bovine factor H were investigated with respect to the sensitivity to limited trypsin digestion. The high-molecular weight form was cleaved into two fragments with apparent molecular masses of, respectively, 45 kD and 125 kD. The low-molecular weight form was cleaved in a different manner to generate three major fragments with molecular masses of 25 kD, 45 kD and 100 kD, respectively. Limited V8 protease mapping of the two forms yielded similar, yet unidentical, peptide band patterns. Purified bovine factor H appeared to bind agarose-bonded heparin through its anion-binding domain and the binding was inhibited by the presence of free heparin or dextran sulfate.Abbreviations HEPES N-2-hydroxylpiperazine-N-2-ethanesulfonic acid - NP-40 Nonidet P-40 - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TyrS tyrosine-O-sulfate     

Primary structure of the xylose-containingN-linked carbohydrate moiety from ascorbic acid oxidase ofCucurbita pepo medullosa

Ascorbic acid oxidase (E.C.1.10.3.3) from the green zucchini squash (Cucurbita pepo medullosa) is a copper-containing glycoprotein which catalyzes the reaction:l-ascorbic acid +1/2 O₂l-dehydroascorbic acid + H₂O. The carbohydrate content of the purified plant glycoprotein amounted to 3% (w/w), and monosaccharide analysis revealed the carbohydrate moiety to be of theN-glycosidic type. The carbohydrate chains were released from the apoenzyme by digestion with PNGase-F immobilized on Sepharose 4B. After fractionation on Bio-Gel P-2 and purification on Mono-Q, the neutral oligosaccharide was investigated by 500-MHz¹H-NMR spectroscopy. The primary structure of theN-linked carbohydrate chain was established to be: Abbreviations AAO ascorbic acid oxidase - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - Man mannose - Xyl xylose - GLC gas-liquid chromatography - FPLC fast protein liquid chromatography - NMR nuclear magnetic resonance - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The polypeptide structure and assembly of Ly-2/3 heterodimers

Mild reduction of mature, thymic Ly-2/3 heterodimers of M _r 67 000 resulted in dissociation into three individual polypeptide chains, , , and , of respective M _r values 38000, 35000, and 30000. The and chains were both immunoprecipitated by a monoclonal antibody directed to the Ly-2.1 epitope whereas the Ly-3.1 antibody bound only the chain. The possibility that the and chains of each heterodimer established their interchain links within a labile precursor protein in which a and segments were fused was considered but discounted by the finding that in mice heterozygous for both Ly-2 and Ly-3 loci, the Ly-2 product of one chromosome was not exclusively joined to Ly-3 structures coded by the same chromosome. By utilizing ionic detergents which selectively alter the charge of intrinsic membrane proteins, both Ly-2 and Ly-3 polypeptides were shown to have membrane insertion sites. It is suggested that as a consequence of their likely synthesis on membrane-bound polysomes, newly synthesized Ly-2 and Ly-3 structures accumulate within the same subcellular compartment — the membranes of the rough endoplasmic reticulum. Their elevated concentration within this space may facilitate a low affinity binding interaction between Ly-2 and Ly-3 which is later stabilized by interchain disulfide bond formation.Abbreviations used in this paper BSA bovine serum albumin - DOC sodium deoxycholate - DTT dithiothreitol - HA hemagglutinin - HTAB hexadecyltrimethylammonium bromide - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TX100 Triton X-100     

Cloning theClostridium thermocellum thermostable laminarinase gene inEscherichia coli: The properties of the enzyme thus produced

Summary We have cloned a 1.9-kb-long fragment ofClostridium thermocellum DNA which encodes laminarinase (EC 3.2.1.39). The enzyme hydrolyzes the -1,3-glucoside bonds in -1,3-and in mixed -1,3-1,4-polyglucans. The enzyme's optimum pH value is around 8.5, temperature optimum –70°C. PAGE-determined mol. weight –32 kDa.Abbreviations used CMC carboxymethyl cellulose - pNPC p-nitrophenyl D cellobioside - pNPLac p-nitrophenyl- D-lactoside - pNPG p-nitrophenyl D glucopyranoside - pNPGal p-nitrophenyl- D galactopyranoside - pNPXyl p-nitrophenyl- - D xylopyranoside - Ap ampicillin - SDS-PAGE SDS polyacrylamide gel electrophoresis     

Primary structural sequence polymorphism in the human class II MHC antigen 33.1

Monoclonal antibody 33.1 defines a non-DR, class 11, human major histocompatibility complex antigen, 33.1, which appears to be distinct from other class II antigens in its cellular distribution and primary structure. To characterize the structure more fully and to determine the degree of polymorphism within 33.1, a comparative N-terminal sequence study has been undertaken using a series of ten B lymphoblastoid cell lines with different DR and MB types. The results confirm that both the and chains of 33.1 are homologues of the corresponding chains of the murine I-A antigen and indicate that while 33.1 does not appear to be identical with MB, it is closely related. Sequence analyses revealed two major variants of 33.1, corresponding to cells with specificities MB1 and MB 3, respectively. Within each MB type, other polymorphisms have been detected. Cells that are MB2 do not react with monoclonal antibody 33.1. Suggestive evidence is presented that monoclonal antibody 33.1 reacts predominantly with the chain of the antigen. The preferential expression of 33.1 on activated B cells suggests that expression of at least the 33.1 chain gene is greatly enhanced in the course of B-cell activation, but the specific function of 33.1 remains to be determined.Abbreviations used in this paper McAb monoclonal antibody - BLCL B lymphoblastoid cell lines - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - NP-40 Nonidet P-40 Fellow of the Arthritis Foundation     

C4 polymorphism in the dog: Molecular heterogeneity of the C4α and C4γ subunit chains

Using an immunoblotting technique and goat antihuman C4, we observed five distinct electrophoretic variants of C4 in a panel of 60 random dogs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated C4 showed that dog C4 is composed of three polypeptide subunit chains (, , and ) and that structural variability occurs within the - and -chain regions. Two distinct molecular weight forms of both the C4- ( _A and _B) and C4-( _A and _B) chain were detected. The variant forms of C4 and C4 were found in association with particular C4 allotypes.     

Genetics of human C4 polymorphism: Detection and segregation of rare and duplicated haplotypes

Applying a combined technology for the detection of allotypec variation of the fourth component of human complement (C4), including immunofixation with anti-C4 and C4-dependent lysis after agarose electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of C4 to separate the C4A and B -chains, and the determination of Rodgers (Rg) and Chido (Ch) determinants of C4 in serum and at the blotted C4 -chains, we detected rare human C4 allotypes and studied the genetic linkage. Partial inhibitors (p. i.) of anti-Rg and anti-Ch sera were found; the C4A51 allotype characterized as Rg p. i. and the C4A1 and C4B51 allotypes as Ch p. i. were genetically inherited. The C4A1 allotype has a unique Rg^- Ch⁺ C4A -chain. Duplicated C4A loci, A ^*3, A ^*2, and A ^*5, A ^*2 were both associated with a C4BQO and the HLA haplotype A3-Cw4-Bw35-DR1. These additions to the already known extensive C4 polymorphism may help to sort out their significance for the biological functions of human C4.Abbreviations used in this paper BF Factor B polymorphism of the alternative pathway of complement activation - C2 second component of complement - C4 fourth component of complement - C4D C4-deficient (C4^*QO/QO) - Ch Chido determinant on C4B^* products - EDTA ethylendiaminetetraacetic acid - GLO I glyoxalase I - HLA human leucocyte antigens, A, B, C and DR (D =related) loci - PAGE polyacrylamide gel electrophoresis - PGM3 phosphoglucomutase, third locus - p. i. partial inhibitor = serological inhibition of some, but not all anti-Ch and anti-Rg sera at selected dilutions - SDS sodium dodecyl sulphate; 94k/96k, 94 000 and 96 000 dalton molecular weight Presented in part at the 1V International Workshop on the Genetics of Complement, July 13–15, 1982, Boston, MA, and the Xth International Complement Workshop, May 25–27,1983 in Mainz, Federal Republic of Germany.     

Aspartic proteinase from wheat seeds: isolation,properties and action on gliadin

Wheat endosperm was shown to contain an aspartic proteinase capable of hydrolyzing the wheat storage protein, gliadin, in vitro. The enzyme was purified to homogeneity by affinity chromatography on bacilliquin-silochrome, diethylaminoethyl-Toyopearl ion-exchange chromatography, chromatofocusing, and preparative polyacrylamide gel electrophoresis. The sedimentation constant of the enzyme was 3.4 S and the relative molecular mass (M_r), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 58000 dalton (Da). The purified enzyme was completely inhibited by pepstain whereas other enzyme inhibitors did not affect its activity. The enzyme was found to hydrolyze mainly - and -gliadins with M_r's of 67000–95000 Da, with maximal activity at pH 4.5. The data make it possible to suggest that the enzyme has an endogenous function by initiating proteolysis of storage proteins in germinating wheat seeds.Abbreviations BSA bovine serum albumin - Da dalton - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Regulation of processing of a plant glycoprotein in the Golgi complex: A comparative study usingXenopus oocytes

The synthesis of phytohemagglutinin (PHA), the major seed lectin ofPhaseolus vulgaris, was investigated inXenopus oocytes injected with RNA isolated from developing bean cotyledons. As is the case for normal PHA, oocyte-synthesized PHA polypeptides were found to contain two asparagine-linked oligosaccharide chains, one of which was of the high-mannose type and the other one of the Golgi-modified type, being largely resistant to endo--N-acetylglucosaminidase H digestion and containing fucose. The modified oligosaccharide chain of oocyte-synthesized PHA appeared to be much larger and more heterogeneous with respect to the modified chain normally present on PHA. When the oocytes were injected with purified mRNA for PHA, isolated by hybrid-selection using a PHA complementary-DNA clone, the results were the same as those obtained by injecting total cotyledonary RNA. On the whole, these results indicate that plant glycoproteins are directed to the Golgi complex even when synthesized in an animal cell, and that correct sorting of the oligosaccharide chains to be processed is independent of the cell-type in which protein synthesis occurs. The form of processing is however cell-type specific.Abbreviations endo H endo H--N-acetylglucosaminidase H - ER endoplasmic reticulum - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Morphological and chemical studies on the crystalloid-forming ‘succulent protein’ from normal and ribosome-deficient Aeonium domesticum plastids

Aeonium domesticum cv. variegatum is a mesochimera of the constitution green/white/green with normal proplastids and chloroplasts in the unaffected tissues and ribosome-deficient colourless mutant plastids in the white leaf tissues. All the different plastid types contain succulent protein crystalloids (SPC). For more detailed characterization, the SPC elements were freed from the plastids and purified by gel filtration. Electron microscopy of different fractions revealed five levels of structural organization. Beginning with the most complex state, the levels are designated as succulent protein (SP) organizational state V (hexagonally arranged and closely packed tubules in the stroma of intact plastids) to I (globular protomers of 5 nm diameter as the basic structure of SPCs). Highly purified SP-fractions were shown by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to consist of two or three proteins of M_r 56 kdalton, 58 kdalton and 60 kdalton, depending on the buffer medium used for SP isolation and the duration of storage of leaves in the frozen state. In the urea/SDS-PAGE system, these proteins show similar mobilities to - and -tubulin, but no immunoreaction against antitubulin. The proteolytic cleavage pattern of tubulin subunits and SP proteins are different. Their locations on two-dimensional isoelectric focusing-SDS gels show some overlappings because of microheterogeneities in both proteins in the pH gradient from pH 4.5 to 6.5. Malatedehydrogenase activity could not be detected in the purified SP fractions.Abbreviations CAM Crassulacean acid metabolism - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SP succulent protein - SPC succulent protein crystalloid - SPOS succulent protein organizational state     

A high molecular weight terminal pigment (“anchor polypeptide”) and a minor blue polypeptide from phycobilisomes of the cyanobacterium Nostoc sp. (MAC): Isolation and characterization

A 94 kD pigment-polypeptide, which is presumed to be involved in anchoring the phycobilisomes to the thylakoids, was isolated from Nostoc phycobilisomes by gel filtration in 63 mM formic acid. The isolation condition did not require detergents or denaturating reagents, as in previous procedures, and enzymatic degradation was not observed at the low pH of 2.5. The anchor polypeptide thus obtained had absorption (Abs) and fluorescence maxima (Em) at 658 and 673 nm, respectively, in 63 mM formic acid at room temperature. The maxima shifted to longer wavelengths in 100 mM potassium phosphate (pH 6.8), Abs 665 and Em 683 nm at room temperature, and Abs 665 and Em 684 nm at liquid nitrogen temperature. The fluorescence maxima at both temperatures correspond to the longest wavelength component resolved in phycobilisomes from second derivative spectra. A minor blue polypeptide was also found by this isolation method. The molecular weight of this polypeptide was ca. 18,000 and is probably similar to a polypeptide which has been found in the phycobilisome core of other cyanobacteria.Abbreviations used -APB -subunit of allophycocyanin B - APC allophycocyanin - kD kilodalton - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - PBS phycobilisome - PE phycoerythrin - PC phycocyanin - PS pigment system     

γ-Tubulin is a component of the Spitzenkörper and centrosomes in hyphal-tip cells ofAllomyces macrogynus

Summary A monoclonal antibody was used to localize -tubulin in hyphal tip cells of the chytridiomycete fungusAllomyces macrogynus, and its distribution determined with standard epifluorescence and laser scanning confocal microscopy. The results demonstrate that -tubulin is a component of the Spitzenkörper and centrosomes. Immunoblot analysis of total soluble protein extracts separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified a single 56 kDa -tubulin-related polypeptide. Localization of -tubulin to the Spitzenkörper ofA. macrogynus provides evidence that the Spitzenkörper in this fungus functions as a microtubule-organizing center.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole - DIC differential interference contrast - LSCM laser scanning confocal microscopy - MTOCs microtubule-organizing centers - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SPB spindle pole body - YpSs yeast extract-inorganic phosphate-soluble starch     

Monoclonal antibodies to the major structural glycoprotein of the Chlamydomonas cell wall

A family of monoclonal antibodies (McAb) has been raised to the major cell-wall structural glycoprotein of the green alga Chlamydomonas reinhardii. The work represents an approach using McAb to the structure and function of plant cell wall-components. On the basis of cross-competition assays, the McAb have been subdivided into six groups. Various lines of evidence indicate that some of these group's e.g. group III, contain McAb which recognise specific oligosaccharide side chains of the glycoprotein. Heterogeneity of the oligosaccharide side chains is demonstrated, by probing Western blots of separated glycoproteins and glycopeptides with the McAb. The results are discussed in relation to the possibility of raising McAb as probes for the structure and function of higher-plant cell-wall oligosaccharides, which are increasingly being shown to be important in cell-cell recognition phenomena and host-pathogen interactions.Abbreviations McAb monoclonal antibody (bodies) - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Partial purification of myosin from lily pollen tubes by monitoring with in vitro motility assay

Summary Myosin in pollen tubes ofLilium longiflorum was partially purified, using an in vitro motility assay as a monitor. The main components in the partially purified preparation had molecular masses of 110, 120, and 140 kDa in SDS-PAGE. They became bound to actin filaments in an ATP-dependent manner. Among the components, only that of 120 kDa became bound to ATP and was concluded to be the heavy chain of pollen tube myosin.Abbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EB extraction buffer - EGTA ethyleneglycol-bis-(-aminoethylether) N, N, N, N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate - TBS Tris buffered saline - TEB Tris-EGTA buffer     

The presence of 17K Mr protein,a major specific substrate for kinase C,found in the Triton-insoluble fraction of synaptosome prepared from rat brain

Cytoskeletal preparation obtained from synaptosome fractions of rat cerebrum contained the activity of kinase C, which phosphorylated 17K Mr protein endogenous to the preparation. The kinase C activity associated with the synaptosome cytoskeletons is greater in the cerebellum and hippocampus than in the cerebrum. The enhancement rates of phosphorylation of the 17K Mr protein were 293%, 544%, and 526% in the Triton X-100-insoluble fractions of synaptosomes prepared from cerebral cortex, hippocampus, and cerebellum, respectively. The 17K Mr protein was distinct from myelin basic protein (MBP) for the following reasons: 1) The electrophoretic mobility of the protein was slightly smaller than that of major MBP of rat in the polyacrylamide gel of 10–20% linear gradient, and the protein was not contained in the purified rat myelin. 2) The isoelectric point of the protein was in neutral range, whereas that of MBP was in alkaline one. 3) The 17K Mr protein did not cross-react with anti-MBP antibody. The protein was shown to be a major substrate contained in the cytoskeletal preparation of synaptosome obtained from cerebrum except for contaminating MBP. Only serine residue of the 17K Mr protein was phosphorylated by the kinase C endogenous to the preparation. The results suggest strongly that the synaptic role of protein kinase C through phosphorylation of the 17K Mr protein.Abbreviations used EGTA ethyleneglycol-bis(-aminoethyl ether) - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - MBP myelin basic protein - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SPM synaptic plasma membrane     

Cytoskeleton components of inside-out and right-side-out plasma membrane vesicles from plants

Summary Isolated plasma membrane vesicles purified by aqueous polymer two-phase partitioning were used as a model system for studies on the membrane-associated (cortical) cytoskeleton in plants. Actin, as identified by immunoblotting, was found to be specifically attached to plasma membrane vesicles from cauliflower (Brassica oleracea L.). The actin was not washed off as the vesicles were turned inside-out, indicative of a fairly strong attachment. Triton X-100 extraction of plasma membrane vesicles resulted in an insoluble and hence pelletable fraction where actin could be found together with several other proteins. Our results show that the cortical cytoskeleton is to some extent co-purified with the plasma membrane, and we believe that well defined, inside-out and right-side-out plasma membrane vesicles can be used to study the structure and dynamics of the plant cortical cytoskeleton.Abbreviations ATP adenosine 5-triphosphate - BCIP 5-bromo-4-chloro-3-indolyl phosphate - BSA bovine serum albumin - CCD counter-current distribution - DTT dithiothreitol - EDTA ethylene-diamine-tetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether) - GSII 1,3--glucan synthase - HEPES N-[2-hydroxyethyl]-piperazine-N-[2-ethane sulfonic acid] - MES 2-(N-morpholino)ethane sulfonic acid - NBT p-nitro blue tetrazolium chloride - IDP inosine 5-diphosphate - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - PPB potassium phosphate buffer - PM plasma membrane - PMSF phenylmethylsulfonyl fluoride - PVDF polyvinylidene difluoride - PVPP polyvinylpolypyrrolidone - SDS sodium dodecyl sulfate - TBS Tris-buffered saline - TTBS Tris-buffered saline with Tween 20 - Tris tris(hydroxymethyl) aminomethane